This is a renewal application for the Roadmap Initiative (RFA-RM-08-019) 'Membrane Protein Expression Center' (MPEC). The goals are to develop, apply, and disseminate novel expression, purification and crystallization of membrane proteins including hetero-oligomeric membrane protein complexes, targeted toward their structure determination as a means of delineating ligand or protein binding, and function. The focus is toward human health, hence on methods appropriate for eukaryotic membrane proteins that generally require eukaryotic expression systems, alongside the relatively simpler bacterial expression of prokaryotic membrane proteins appropriate sometimes to homologs. New initiatives currently in development are: (1) principles of gene redesign to optimize expression. (2) a new cell free expression method from yeast, a eukaryotic source. (3) cell free synthesis in the context of lipids that can proceed directly to mesophase crystallization. (4) presentation of membrane proteins for optimization of Fab fragments as crystallization chaperones. (5) means of expressing partners and assembling functional membrane protein heterooligomeric complexes in a robust yeast expression system. (6) expression in a human HEKs cells mutated to minimize heterogeneity in post translational modification. (7) identifying partners in functional hetero-oligomeric complexes of 500 yeast membrane proteins (8) membrane protein purification. (9) microfluidic applications of lipidic sponge phase crystallization, and crystallization in microfluidic chips that can be directly inserted into the X-ray beam for screening by X-rays. (10) Target selection based on value to human health. The expression systems harness a suite of Ligase Independent Cloning (LIC) vectors developed at MPEC for insertion of genes into specially adapted forms of S. cerevisiae, Pichia pastoris, or HEKs 293 cells. The focus toward human health is reflected in the choice of human, pathogen, occasionally prokaryotic homologs, and other high value targets supported by Principal Investigators and collaborators with experience in the structural and functional biology of particular membrane protein classes. Thus identifying the most important targets and providing vision and focus for high impact in each area. These areas include channels, ion channels, transporters, secondary transporters, proteins of bioenergetic importance, G Protein Coupled Receptors, neuroreceptors, nuclear pores, intracellular receptors including that for the unfolded protein response. PUBLIC HEALTH RELEVANCE: Membrane proteins constitute ~30% of all eukaryotic proteins and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. This proposal seeks to overcome roadblocks to expression, purification and structural determination of membrane proteins in general, and targets of importance to human health in particular. Functional assays will serve to assist in discovery of therapeutics and understanding function.